This invention relates to polynucleotide sequences encoding fusion proteins, for use in gene therapy in particular for Parkinson""s disease. The invention also relates to vectors carrying the polynucleotide sequences, in particular retroviral vectors, and to their use in gene therapy.
Parkinson""s disease (PD) is a common neurodegenerative disorder that afflicts the growing population of elderly people. Patients display tremor, cogwheel rigidity and impairment of movement. It is generally thought to be an acquired rather than inherited disease in which environmental toxins, metabolic disorders, infectious agents and normal ageing have all been implicated. PD is associated with the degeneration of nigrostriatal neurons which have their soma located in the substantia nigra. They send axonal projections to the basal ganglia and they use dopamine as their neurotransmitter. Some features of the disease can be controlled by the administration of L-DOPA, the metabolic precursor to dopamine, which diffuses across the blood brain barrier more effectively than dopamine itself. Unfortunately as the disease progresses the side effects of this treatment become unacceptable.
PD is an ideal candidate for gene therapy for several reasons. The clinical efficacy of systemic administration of L-DOPA suggests that restoration of neuronal circuitry is not essential for disease management. Therefore genetic manipulation of brain cells to provide local production of L-DOPA from tyrosine may be a realistic strategy for treatment. The biosynthesis of L-DOPA from tyrosine involves a single step suggesting that provision of tyrosine hydroxylase (TH) by genetic means may be sufficient and some success has been achieved using this strategy in small animals and in cell culture (Kaplitt et al., 1994 Nature Genetics 8, 148; During et al., 1994 Science 266, 1399; Horellou et al., 1994 Neuroreport 6, 49; Owens et al., 1991 J. Neurochem. 56, 1030). However, if one is to use local endogenous brain cells as L-DOPA factories for the treatment of PD in man it is likely that high levels of L-DOPA will be required to effect a treatment. These high levels must be efficiently converted to dopamine as the necessary neurotransmitter and primary therapeutic agent. It is likely therefore that it will be necessary not only to supply tyrosine hydroxylase but also DOPA decarboxylase (DD), the enzyme that converts L-DOPA to dopamine. This means that in a gene therapy strategy the genes for both of these enzymes will be required.
Amongst gene transfer systems retroviral vectors hold substantial promise for gene therapy. These systems can transfer genes efficiently and new vectors are emerging that are particularly useful for gene delivery to brain cells (Naldini et al., 1996 Science 272, 263). However, it is clear from the literature that retroviral vectors achieve the highest titres and most potent gene expression properties if they are kept genetically simple (PCT/GB96/01230; Bowtell et al., 1988 J.Virol. 62, 2464; Correll et al., 1994 Blood 84, 1812; Emerman and Temin 1984 Cell 39, 459; Ghattas et al., 1991 Mol.Cell.Biol. 11, 5848; Hantzopoulos et al., 1989 PNAS 86, 3519; Hatzoglou et al., 1991 J.Biol.Chem 266, 8416; Hatzoglou et al., 1988 J.Biol.Chem 263,17798; Li et al., 1992 Hum.Gen.Ther. 3, 381; McLachlin et al., 1993 Virol. 195, 1; Overell et al., 1988 Mol.Cell Biol. 8, 1803; Scharfman et al., 1991 PNAS 88, 4626; Vile et al., 1994 Gene Ther 1, 307; Xu et al., 1989 Virol. 171, 331; Yee et al., 1987 PNAS 84, 5197). This means only using a single transcription unit within the vector genome and orchestrating appropriate gene expression from sequences within the 5xe2x80x2 LTR. If there is a need to express two enzymes, such as TH and DD, from a single retroviral vector the only solution would be to use an internal ribosome entry site (IRES) to initiate translation of the second coding sequence in a polycistronic message (Adam et al 1991 J.Virol. 65, 4985). However, the efficiency of an IRES is often low and tissue dependent making this strategy undesirable when one is seeking to maximise the efficiency of metabolic conversion of tyrosine through to dopamine. The present invention addresses these problems.
The present invention provides in one aspect a polynucleotide sequence for use in gene therapy, which polynucleotide sequence comprises two or more therapeutic genes operably linked to a promoter, and encodes a fusion protein product of the therapeutic genes. The invention thus provides a way of expressing two therapeutic genes from a single xe2x80x9cchimeric genexe2x80x9d.
In another aspect, the invention provides a vector carrying the polynucleotide sequence as described. The vector may be for example an expression vector such as a plasmid, or it may be a retroviral vector particle comprising an RNA genome containing the polynucleotide sequence as described herein.
In yet further aspects, the invention provides a DNA construct encoding the RNA genome for the retroviral vector particle; and a retroviral vector production system comprising a set of nucleic acid sequences encoding the components of the retroviral vector particle.
The invention further provides the use of retroviral vectors carrying the chimeric gene described herein, in gene therapy and in the preparation of a medicament for gene therapy; and a method of performing gene therapy on a target cell, which method comprises infecting and transducing the target cell using a retroviral vector particle as described herein. The invention further provides transduced target cells resulting from these methods and uses. The invention thus provides a gene delivery system for use in medicine.
In addition, the invention provides a polynucleotide sequence encoding a fusion protein comprising tyrosine hydroxylase and DOPA decarboxylase in either TH-DD or DD-TH order, linked by a flexible linker.
The therapeutic genes are chosen according to the effect sought to be achieved. The fusion protein has or is capable of having the desired activity of the therapeutic gene products. The product encoded by one or more of the therapeutic genes may be an enzyme. The fusion protein may thus display the activity of one or more enzymes. Where the therapeutic genes encode two different enzymes, the resulting fusion protein is a bifunctional enzyme. In the specific example described herein, the fusion protein comprises the enzymes tyrosine hydroxylase and DOPA dehydroxylase having enzyme activities as described above.
Preferably the therapeutic genes are linked by a sequence encoding a flexible linker. A suitable linker may comprise amino acid repeats such as glycine-serine repeats. The purpose of the linker is to allow the correct formation and/or functioning of the therapeutic gene products. It must be sufficiently flexible and sufficiently long to achieve that purpose. Where the therapeutic genes encode two different enzymes, the linker needs to be chosen to allow the functioning of both of the enzymes. The coding sequence of the flexible linker may be chosen such that it encourages translational pausing and therefore independent folding of the protein products of the therapeutic genes.
A person skilled in the art will be able to design suitable linkers in accordance with the invention. Some specific examples of suitable linkers are given below; it will be evident that the invention is not limited to these particular linkers.
1. (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 21) as described in Somia et al., 1993 PNAS 90, 7889.
2. (Gly-Gly-Gly-Gly-Ser)5 (SEQ ID NO: 22), a novel linker.
3. (Asn-Phe-Ile-Arg-Gly-Arg-Glu-Asp-Leu-Leu-Glu-Lys-Ile-Ile-Arg-Gln-Lys-Gly-Ser-Ser-Asn) (SEQ ID NO: 23) from HSF-1 of yeast, see Wiederrecht et al., 1988 Cell 54, 841.
4. (Asn-Leu-Ser-Ser-Asp-Ser-Ser-Leu-Ser-Ser-Pro-Ser-Ala-Leu-Asn-Ser-Pro-Gly-Ile-Glu-Gly-Leu-Ser) (SEQ ID NO: 24) from POU-specific OCT-1, see Dekker et al., 1993 Nature 362, 852 and Sturm et al., 1988 Genes and Dev. 2, 1582.
5. (Gln-Gly-Ala-Thr-Phe-Ala-Leu-Arg-Gly-Asp-Asn-Pro-Gln-Gly) (SEQ ID NO: 25) from RGD-containing Laminin peptide, see Aumailly et al., 1990 FEBS Lett. 262, 82.
6. (Ser-Gly-Gly-Gly-Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr-Gly-Gly-Ser-Ser-Pro-Gly) (SEQ ID NO: 26) from LDV-containing linker, see Wickham et al., Gene Therapy 1995 2, 750.
It will be evident that the term xe2x80x9cgenexe2x80x9d is used loosely here, and includes any nucleic acid coding for the desired polypeptide.
Vectors carrying the polynucleotide sequence encoding the fusion protein include any vectors suitable for use in gene therapy, that is, vectors which are capable of delivering the polynucleotide sequence encoding the fusion protein to a target cell. Particularly preferred vectors for use in the present invention are retroviral vectors, which have a number of well-documented advantages as vectors for gene therapy. The invention provides retroviral vectors with further advantages for gene therapy, because it enables the construction of a simplified retroviral vector for delivering therapeutic genes. In particular it enables the construction of retroviral vectors capable of delivering multiple therapeutic genes in a single transcription unit. Multiple therapeutic genes in a single transcription unit are operably linked to the same promoter and are all transcribed under the control of that promoter. There is no more than one promoter active in respect of the single transcription unit at any one time.
Preferably, the retroviral vector according to the invention is a single transcription unit vector, that is, the vector genome in DNA or RNA form is under the transcriptional control of no more than one vector promoter at any one time. In a preferred embodiment, this is achieved by locating the polynucleotide sequence according to the invention such that in the DNA form of the vector genome integrated into the target cell genome (the DNA provirus), it is under transcriptional control of the 5xe2x80x2 LTR. There are alternative ways of achieving a single transcription unit vector, however. The vector genome could be designed as a self-inactivating vector (Yu et al., 1986 PNAS 83, 3194) in which part of the 3xe2x80x2 U3 sequences are deleted so that the transduced vector genome has a non-functional 5xe2x80x2 LTR promoter. The polynucleotide sequence according to the invention would be operably linked to an internal conditional promoter between the LTRs which could be activated once the vector has transduced a target cell. Activation of the promoter might be dependent upon cellular or external factors.
Although single transcription unit vectors are preferred, other vectors are not excluded. It may be useful for example to include a marker gene in the vector, operably linked to a different promoter which may be active simultaneously with the promoter responsible for transcription of the polynucleotide sequence encoding the fusion protein. A marker gene encoding a selectable marker may be useful for selecting successfully transfected packaging cells, or successfully transduced target cells. Marker genes encoding selectable markers may be for instance drug resistance genes or metabolic enzyme genes.
The retroviral vector according to the invention may be constructed according to methods known in the art. Retroviral vectors suitable for gene therapy will need to be replication defective. Particular factors to be taken into consideration when constructing a retroviral vector include safety aspects and the avoidance of undesirable immune responses. Preferably, the retroviral vector genome which will be inserted into the target cell in the form of a DNA provirus contains the minimum retroviral material necessary to function. This avoids both the possible reconstruction of infectious virus particles, and expression of unwanted virus proteins in the target cell which could otherwise evoke undesirable immune responses in the patient being treated.
For the production of retroviral vector particles, the vector RNA genome is expressed from a DNA construct encoding it, in a host cell. The components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the xe2x80x9cpackaging systemxe2x80x9d, which usually includes either or both of the gag,pol and env genes) expressed in the host cell. The set of sequences required for the production of the retroviral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.
To date, the most widely used retroviral vector systems for human gene therapy applications have used MLV. However, retroviral vector systems may also be based on other oncoretroviruses (the sub-group of retroviruses containing MLV), or lentiviruses (the subgroup of retroviruses containing HIV, SIV, FIV, BLV, EIAV, CEV and visna virus), or retroviruses from other subgroups. A range of retroviruses have already been split into packaging and vector components for retroviral vector particle production systems, including ASLV, SNV and RSV. It will be evident that a retroviral vector according to the invention need not be confined to the components of a particular retrovirus. The retroviral vector may comprise components derived from two or more different retroviruses, and may also comprise synthetic components. Vector components can be manipulated to obtain desired characteristics, such as target cell specificity.
Certain retroviruses have special characteristics which may be useful in particular gene therapy applications. For example, the lentiviruses such as HIV are capable of infecting and transducing non-dividing cells because they have means for getting the proviral DNA across the nuclear membrane of target cells. This feature will be useful if it is desired to target non- or slowly-dividing cell types in gene therapy. Such cell types include the neurons of the human brain, which are a potentially important target for gene therapy treatment of Parkinson""s disease. A retroviral vector particle according to the invention may thus be derived from a lentivirus, at least to the extent that it is capable of delivering proviral DNA efficiently to a non-dividing or slowly-dividing cell.